Tumor necrosis factors are a class of proteins produced by numerous cell-types, including monocytes and macrophages. At least two TNFs have been previously described, specifically TNF alpha and TNF beta (lymphotoxin).
These known TNFs have important physiological effects on a number of different target cells involved in the inflammatory response. The proteins cause both fibroblasts and synovial cells to secrete latent collagenase and prostaglandin E2, and cause osteoblastic cells to carry out bone resorption. These proteins increase the surface adhesive properties of endothelial cells for neutrophils. They also cause endothelial cells to secrete coagulant activity and reduce their ability to lyse clots. In addition they redirect the activity of adipocytes away from the storage of lipids by inhibiting expression of the enzyme lipoprotein lipase. TNFs cause hepatocytes to synthesize a class of proteins know as xe2x80x9cacute phase reactantsxe2x80x9d and they act on the hypothalamus as pyrogens. Through these activities, it has been seen that TNFs play an important part in an organism""s response to stress, to infection, and to injury. See, e.g., articles by P. J. Selby et al. in Lancet, Feb. 27, 1988, pg. 483; H. F. Starnes, Jr. et al. in J. Clin. Invest. 82: 1321 (1988); A. Oliff et al. in Cell 50:555 (1987); and A. Waage et al. in Lancet, Feb. 14, 1987, pg. 355.
However, despite their normally beneficial effects, circumstances have come to light in which the actions of TNFs are harmful. For example, TNF alpha injected into animals gives rise to the symptoms of septic shock; endogenous TNF levels have been observed to increase following injection of bacteria or bacterial cell walls. TNFs also cause bowel necrosis and acute lung injury, and they stimulate the catabolism of muscle protein. In addition, the ability of TNFs to increase the level of collagenase in an arthritic joint and to direct the chemotaxis and migration of leukocytes and lymphocytes may also be responsible for the degradation of cartilage and the proliferation of the synovial tissue in this disease. Therefore, TNFs may serve as mediators of both the acute and chronic stages of immunopathology in rheumatoid arthritis. TNFs may also be responsible for some disorders of blood clotting through altering endothelial cell function. Moreover, excessive TNF production has been demonstrated in patients with AIDS and may be responsible for some of the fever, acute phase response and cachexia seen with this disease and with leukemias.
In these and other circumstances in which TNF has a harmful effect, there is clearly a clinical use for an inhibitor of TNF action. Systemically administered, TNF inhibitors would be useful therapeutics against septic shock and cachexia. Locally applied, such TNF inhibitors would serve to prevent tissue destruction in an inflamed joint and other sites of inflammation. Indeed, such TNF inhibitors could be even more effective when administered in conjunction with interleukin-I (IL-1) inhibitors.
One possibility for therapeutic intervention against the action of TNF is at the level of the target cell""s response to the protein. TNF appears to act on cells through a classical receptor-mediated pathway. Thus, any molecule which interferes with the ability of TNF to bind to its receptors either by blocking the receptor or by blocking the TNF would regulate TNF action. For these reasons, proteins and small molecules capable of inhibiting TNF in this manner have been sought by the present inventors.
As noted above, this invention relates to TNF inhibitors generally, and, more specifically, to a urine-derived TNF inhibitor. Additionally, the present invention relates to biologically-active analogs of this inhibitor.
An object of the present invention is to provide purified forms of TNF inhibitor which are active against TNF alpha. An additional object of the present invention is to provide these inhibitors in purified forms to enable the determination of their amino acid sequence. A further object is to provide the amino acid sequences of certain TNF inhibitors. In addition it is an object of this invention to provide a cellular source of the mRNA coding for TNF inhibitors and a cDNA library containing a cDNA for the inhibitors. Furthermore, it is an object of this invention to provide a genomic clone of DNA coding for the TNF inhibitors, and the coding sequences of that DNA.
The identification of biologically-active analogs of such TNF inhibitors with enhanced or equivalent properties is also one of the objects of the invention.
Additionally, it is an object of this invention to provide a recombinant-DNA system for the production of the TNF inhibitor described herein. A further object of the present invention includes providing purified forms of TNF inhibitor which would be valuable as pharmaceutical preparations exhibiting activity against TNF. Another object of the present invention includes providing purified combinations of TNF inhibitors and IL-1 inhibitors which are valuable as pharmaceutical preparations exhibiting activity against both IL-1 and TNF.
The inventors of the present invention have isolated at least two TNF inhibitor proteins with TNF-inhibiting properties. A 30kDa protein and a 40kDa protein have been obtained in their purified forms. The amino acid sequence of the 30kDa TNF inhibitor protein has been obtained. The amino acid sequence data of the 40kDa TNF inhibitor protein has also been obtained. Both the 30kDa TNF inhibitor and the 40kDa TNF inhibitor are novel, previously undescribed proteins.
A human genomic DNA clone which contains the gene for the 30kDa protein has been obtained. A cell source of this protein has been identified and a cDNA clone has been obtained and the nucleic acid sequence of the gene for the protein determined. In addition, the gene clone has been placed in a vector which has been found to express the protein in host cells. Also a process has been developed for purifying the protein in an active form.
A cell source has been identified which produces the 40kDa protein and a cDNA clone has been obtained and the nucleic acid sequence determined of the gene for the 40kDa protein. The full cDNA clones encoding for both the 30kDa TNF inhibitor precursor and the 40kDa TNF inhibitor precursor have been expressed in mammalian cells to yield an increase in TNF binding sites on the cell surface.
A gene coding for the mature form of the 30kDa protein has been expressed in E. Coli. Three seperate genes coding for all or portions of the mature 40kDa protein have also been expressed in E. Coli. The three 40kDa Inhibitor proteins expressedxe2x80x94mature 40kDa TNF inhibitor, 40kDa TNF inhibitor xcex9451 and 40kDa TNF inhibitor xcex9453xe2x80x94each exhibit TNF inhibiting activity. Mature 40kDa TNF inhibitor, as isolated from medium conditioned by human U937 cells, and 40kDa TNF inhibitor xcex9451 and 40kDa TNF inhibitor xcex9453, are collectively referred to as 40kDa TNF inhibitor.
The 30kDa TNF inhibitor has shown activity in inhibiting TNF alpha. The 40kDa TNF inhibitor has shown inhibitory action against both TNF alpha and TNF beta.
It will now be possible to perform the large scale production of these TNF inhibitors through recombinant DNA technology. These inhibitors should be suitable for use in pharmaceutical formulations useful in treating pathophysiological conditions mediated by TNF.